Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
  • G01N33/492—Determining multiple analytes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
  • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes

Definitions

• the present invention is related to the field of electrochemical sensors, particularly
• characteristics of the patient's blood such as pH, hematocrit, the ion concentration of calcium,
• electrochemical sensor systems which provide blood chemistry analysis are
• blood source such as a heart/lung machine used to sustain a patient during surgery.
• small test samples of ex vivo blood can be diverted off-line from either the venous or arterial flow lines of a heart/lung machine directly to a chamber exposed to a bank of micro-
• Electrochemical sensor systems are analytical tools combining a chemical or biochemical
• recognition component e.g., an enzyme
• a physical transducer such as a platinum electrode
• the chemical or biochemical recognition component is capable of selectively interacting with an analyte of interest and of generating, directly or indirectly, an electrical signal through the
• Electrochemical sensor systems play an increasing role in solving analytical and
• biochemical recognition compounds is also compromised by residual levels of analyte remaining
• One objective of the present invention is to provide a system and method for increasing
• This inner polymeric membrane functions to protect
• the electrochemical sensor from the fouling or interference by compounds in the sample and thus
• an electrochemical sensor includes at least one
• the composite membrane includes an outer layer, an outer layer, and an outer layer, and an outer layer, and an outer layer, and an outer layer.
• the enzyme layer and a restorable inner layer.
• the inner layer is in contact with at least one
• the outer layer of the composite membrane may include a compound selected from the
• enzyme layer of the electrochemical sensor includes a H 2 O 2 generating enzyme, such as glucose
• the enzyme layer includes one
• glucose oxidase or a combination of several enzymes, such as a mixture of glucose oxidase, lactate oxidase,
• the electrochemical sensor h one embodiment, the electrochemical sensor
• the inner layer of the electrochemical sensor may include a compound
• an electrochemical sensor cartridge includes an
• electrochemical sensor card at least one electrochemical sensor, and a reservoir containing an
• electropolymerizable monomer solution in fluid communication with the electrochemical sensor
• the electrochemical sensor cartridge may
• an electrochemical sensor card that includes at least one composite membrane, h
• the electrochemical sensor cartridge may include a composite membrane
• the electrochemical sensor cartridge includes
• At least one calibration solution reservoir in fluid communication with the electrochemical sensor
• electropolymerizable monomer solution may be combined with
• the calibration solution in a single reservoir.
• electrochemical sensor cartridge includes electropolymerizable monomer solution in the calibration solution wherem the concentration of the monomer is in the range of about 1-100 mM.
• at least one of the electrochemical sensors of the electrochemical sensor cartridge comprises an enzyme electrode sensor
• the electrochemical sensor of the electrochemical sensor cartridge is formed on an electrode composed from a material selected from a group consisting of platinum, gold, carbon or one of their modified structure
• the electrochemical sensor includes an electropolymerizable monomer selected from a group consisting of benzothiophene, phenylenediamines, and dihydroxybenzenes.
• the electrochemical sensor is selective for a hydrogen ion, carbon dioxide, oxygen, sodium ion, potassium ion, ionized calcium, chloride, hematocrit, glucose, lactate, creatine, creatinine or urea, h yet another embodiment, the electrochemical sensor includes a electropolymerizable monomer that is a derivative of phenylenediamine.
• an electrochemical sensor system in another aspect of the present invention, includes an electrochemical sensor card including at least one electrochemical sensor, wherein the electrochemical sensor includes at least one polymeric membrane.
• the electrochemical sensor system also includes an electrochemical sensor apparatus that is in electrical contact with the electrochemical sensor card.
• the electrochemical sensor apparatus is configured to measure electrical signals from the electrochemical sensor card and is capable of providing an electrical potential to the electrochemical sensor for the polymerization of the electropolymerizable monomer solution to the polymeric membrane.
• the electrochemical sensor system also includes a reservoir containing an electropolymerizable monomer solution in fluid communication with the electrochemical sensor card. The electropolymerizable monomer solution is polymerized to the polymeric membrane by the electrical potential provided by the electrochemical sensor apparatus.
• the electrochemical sensor cartridge may
• an electrochemical sensor card that includes at least one composite membrane, hi
• the electrochemical sensor cartridge may include a composite membrane
• the electrochemical sensor system further includes a calibration
• concentration of the electropolymerizable monomer solution is in the range of about 1-lOOmM.
• the electrochemical sensor system includes at least one enzyme electrode
• the electrochemical sensor system includes an
• electrochemical sensor that is selective for a compound selected from a group consisting of
• hematocrit glucose, lactate, creatine, creatinine or urea.
• the electrochemical sensor system includes an
• electropolymerizable monomer that is selected from a group consisting of benzothiophene,
• electropolymerizable monomer solution in the calibration solution is 1-100 mM.
• electrochemical sensor system includes an electrochemical sensor apparatus
• electrochemical sensor system further comprises
• the invention relates to accelerating the recovery of the electrochemical
• interfering agents after exposure of the electrochemical sensor to a sample, are examples of interfering agents.
• interfering agents is the residual concentration of the electropolymerizable
• an electrochemical sensor including an electrode and a composite membrane
• composite membrane including at least one polymeric membrane, an electrical source in
• the electrical potential is in a
• the electrical potential is about 0.4
• N versus the on-board reference electrode and is applied for about 50 seconds.
• the invention relates to the method of restoring the functional properties
• the method includes providing an electrochemical system, which
• the electrochemical sensor card includes at least one electrochemical sensor.
• electrochemical sensor includes an electrode and a composite membrane, the composite
• the electrochemical sensor system also includes
• the electrochemical sensor apparatus includes an electrochemical sensor apparatus in electrical contact with the electrochemical sensor card.
• the electrochemical sensor apparatus is configured to measure electrical signals from the
• electrochemical sensor card and to provide an electrical potential to the electrochemical sensor.
• the electrochemical sensor system also includes a reservoir containing an electropolymerizable
• electropolymerizable monomer solution is polymerized to the polymeric membrane by the
• the functional properties of an electrochemical sensor also includes contacting the electrochemical sensor with the solution and applying an electrical potential of sufficient strength
• the senor includes adding the electropolymerizable monomer to a calibrating solution to form the
• the electrical potential comprises a
• the electrical potential comprises
• sensor further includes the step of applying an additional electrical potential of sufficient strength
• the electrical potential is in a range of about 0.1
• the invention relates to the method for restoring the functional properties
• the method includes the steps of connecting an electrochemical sensor cartridge that includes an electrochemical sensor to an electrochemical
• the electrochemical sensor includes an electrode and a composite membrane
• the method further includes contacting the
• the method further includes adding an electropolymerizable
• an electrical potential is applied at a range of about 0.1 to 0.8 V versus
• the electrical potential may be applied for a range of time
• the method also includes applying an
• the electrical potential is in a range of about 0.1 to 0.8 V versus the on-board
• reference electrode is applied for a range of time from about 10 to 200 seconds.
• the invention in another aspect, relates to a composite membrane for a biosensor.
• biosensor includes an inner membrane layer, an outer membrane layer, and an enzyme layer.
• enzyme layer includes a matrix that includes at least one enzyme, a cross-linking agent, and an
• the composite membrane is formed from
• lactate oxidase includes one or more of the enzymes lactate oxidase, creatinase, sarcosine oxidase, and creatininase.
• the invention in another aspect, relates to a matrix for an enzyme sensor.
• the matrix for an enzyme sensor.
• lactate oxidase includes lactate oxidase, a cross-linking agent, and a enzyme stabilizer.
• the enzyme stabilizer includes lactate oxidase, a cross-linking agent, and a enzyme stabilizer.
• the matrix forms a cross-linked matrix of proteins having enzymatic activity.
• the matrix may form an electrochemical electrode.
• the matrix may also include bovine serum albumin. Other inert proteins similar to bovine serum albumin may also be included.
• one or more of the cross-linking agent present in the matrix may include a dialdehyde, glutaraldehyde, for example, a diisocyanato, 1,4-diisocyanatobutane, for example, and a diepoxide, 1,2,7,8- diepoxyoctane and 1,2,9,10-diepoxydecane, as examples, hi another embodiment, the cross- linking agent present in the matrix isl-10% glutaraldehyde by weight, hi yet another embodiment, the cross-linking agent present in the matrix is5% glutaraldehyde by weight, h another embodiment, the enzyme stabilizer present in the matrix may include one or more of the compounds, polyethyleneimine, polypropyleneimine, poly(N-vin
• the invention relates to a matrix for an enzyme sensor that includes creatinase, sarcosine oxidase, a cross-linking agent and, an enzyme stabilizer, hi one embodiment, the matrix also includes creatininase. In one embodiment, the matrix forms a cross-linked matrix of proteins having enzymatic activity.
• the enzyme sensor may form an electrochemical sensor, h another embodiment, one or more of the cross-linking agent present in the matrix may include a dialdehyde, glutaraldehyde, for example, a diisocyanato, 1,4- diisocyanatobutane, for example, and a diepoxide, 1,2,7,8-diepoxyoctane and 1,2,9,10- diepoxydecane, as examples.
• the cross-linking agent present in the matrix isl-10% glutaraldehyde by weight, h yet another embodiment, the cross-linking agent present in the matrix is5% glutaraldehyde by weight, h another embodiment, the enzyme stabilizer present in the matrix may include one or more of the compounds, polyethyleneimine, polypropyleneimine, poly(N-vinylimidazole), polyallylamine, polyvinylpiridme, polyvinylpyroUidone, polylysine, protamine and their derivatives. In another embodiment, the
• enzyme stabilizer present in the matrix is 1-20% polyethyleneimine by weight.
• the enzyme stabilizer present in the matrix is 5% polyethyleneimine by weight.
• the invention relates to a matrix for an enzyme sensor including one or
• linking agent and an enzyme stabilizer.
• FIG. 1 is a schematic diagram of the components of an electrochemical sensor apparatus
• a sensor cartridge with a bank of sensors and a thermal block for accelerated hydration and calibration of the sensors.
• FIG. 2 illustrates a reverse frontal view of the sensor card, partly fragmentary, of a
• FIGS. 3 A-B illustrate cross-sectional views of an enzyme sensor.
• FIG. 4 illustrates an embodiment of apO 2 sensor.
• FIG. 5 illustrates a frontal view of the electrode card contained in one embodiment of the
• FIG. 6 illustrates cross-sectional views of an ion sensor.
• FIGS. 7A-G illustrate the components of a thermal block assembly.
• the present invention provides electrodes and electrochemical sensor systems for
• aqueous samples including, but not limited to, blood, serum or other
• the invention is directed to such sensors in which the electrodes
• an interference rejection membrane which is the inner polymeric membrane of the
• the invention have increased accuracy and precision and increased effective life spans, hi
• the sensor system is adapted to measure the
• blood gases e.g., oxygen and carbon dioxide
• ions e.g., sodium
• Electrode refers to a component of an electrochemical device
• the internal ionic medium typically, is an aqueous solution with dissolved salts.
• Electrodes are of three types, working or indicator electrodes, reference electrodes, and counter electrodes.
• a working or indicator electrode measures a specific chemical species, such as an ion. When electrical potentials are measured by a working electrode, the method is termed potentiometry. All ion-selective electrodes operate by potentiometry. When current is measured by a working electrode, the method is termed amperometry. Oxygen measurement is carried out by amperometry.
• Working electrodes may also function by including an enzyme as part of an enzyme layer that is part of a composite layer that is in close contact with the electrode.
• the enzyme which is specific to a particular analyte, produces hydrogen peroxide, a by-product of the catalytic reaction of the enzyme on the analyte. Hydrogen peroxide is detected by the electrode and converted to an electrical signal.
• a reference electrode serves as an electrical reference point in an electrochemical device against which electrical potentials are measured and controlled, h one embodiment, silver-silver nitrate forms the reference electrodes. Other types of reference electrodes are mercury-mercurous chloride-potassium chloride or silver-silver chloride-potassium chloride.
• a counter elecfrode acts as a sink for the current path.
• the term "sensor” is a device that responds to variations in the concentration of a given chemical species, such as glucose or lactate, in a sample, such as a body fluid sample.
• An electrochemical sensor is a sensor that operates based on an electrochemical principle and requires at least two electrodes.
• the two elecfrodes include an ion-selective electrode and a reference electrode.
• Amperometric enzyme elecfrodes additionally require a third electrode, a counter electrode.
• enzyme sensors based on two electrodes, a working and reference electrode are also common.
• the term "ion selective electrode” generally refers to a silver wire coated with silver chloride in contact with a buffer solution containing a chloride concenfration (the inner solution).
• the buffer solution is covered with a polymeric ion-selective membrane that is in contact with the test solution.
• the ion selective membrane typically consists of a high
• PVC molecular weight polystyrene resin
• plasticizer an ionophore specific to a particular ion
• borate salt an ionophore specific to a particular ion
• the surface of the polymeric membrane is in contact with the test sample on one side and the
• dry electrochemical sensor refers to the ion selective electrode
• the ion-selective electrodes have the same configuration as described above, however, the inner
• the dried salt must be solubilized in water to obtain a
• enzyme electrode generally refers to a composite membrane
• the composite membrane is
• the middle or enzyme layer comprises at least one protein species
• the enzymatic activity may also be provided by compounds which
• the enzyme is stabilized in a matrix
• the inner or interference rejection membrane is a
• polymeric membrane that functions to insulate the wire electrode from compounds in the sample
• hydrolysis refers to the process of solubilizing the salts of a
• thermal cycling is the process by which the temperature of an
• electrochemical sensor soaked in an aqueous salt solution, is raised to a specified elevated
• characteristics of a sensor to a specific analyte are determined quantitatively.
• the senor is exposed to at least two reagent samples, each reagent sample having a
• the electrochemical sensor system 8 employs a sensor assembly
• samples to be analyzed by the system are introduced through a sample inlet 13 a.
• Blood samples to be analyzed by the system are introduced through a sample inlet 13 a.
• extracorporeal blood flow circuit connected to a patient during, for example, open heart surgery.
• Blood samples may be introduced into the sample inlet 13a through other automatic means, or
• the blood samples may be introduced as discrete samples.
• the electrochemical system 8 including a number of essential components as heretofore
• the electrochemical sensor system 8 incorporates in the cartridge 37 at least two
• prepackaged containers 14, and 16 each containing a calibrating aqueous solution having known
• Calibrating Solution A the prepackaged container 14 contained within the prepackaged container 14
• Calibrating Solution B a solution contained within the prepackaged container 16 will be termed Calibrating Solution B.
• prepackaged containers 14, 16 and 23 contain a sufficient quantity of its calibrating solution to
• the Calibrating Solution AO contains
• Electropolymerizable monomer such as -phenylenediamine
• electropolymerizable monomers is contained in a prepackaged container (not shown) separate
• the prepackaged container 14 is connected to the
• the container 23 is connected to a third input of the multi-position valve 18 through
• Another container 17 contains a rinse solution and is connected to the input of the
• the rinse bag 17 is
• output line 12 is the output of the multi-position valve 18 and is connected to the sample input
• line 12b is open to the sample input line 13b and allows
• the cartridge 37 also includes a container 28, for a reference solution.
• the container 28 is a container 28, for a reference solution.
• the system further includes a container 32
• Both the waste flow conduit 34 and the reference solution flow line 30 consist of or
• the pump includes sections of flexible walled tubing that pass through the peristaltic pump 26.
• the pump includes sections of flexible walled tubing that pass through the peristaltic pump 26.
• Cartridge 37 also contains a sensor card 50 which provides a low volume, gas tight
• the sample such as a blood sample, calibration solution, or monomer-
• reference electrode collectively indicated as sensors 10, are integral parts of the chamber.
• Chemically sensitive, hydrophobic membranes typically formed from polymers, such as
• polyvinyl chloride specific ionophores, and a suitable plasticizer, are permanently bonded to the
• the cartridge 37 also serves as hematocrit, which calibrates at one level.
• the cartridge 37 also serves as hematocrit, which calibrates at one level.
• the cartridge 37 is intended to be discarded and replaced by another
• sensors are available as a bank of elecfrodes 10 fabricated in a plastic
• the thermal block assembly 39 of a suitably adapted blood chemistry analysis machine.
• the thermal block assembly 39 is a suitably adapted blood chemistry analysis machine.
• the heating/cooling devices such as a resistive element or a Peltier-effect device, a
• analog board 45 houses analog-to-digital and digital-to-analog converters. The signal from the
• electrode interface 38 passes through the analog-to-digital converter, converted into digital form
• the polarization voltage for oxygen sensor go through the digital-to-analog
• the electrochemical sensor system 8 is formed upon insertion of the cartridge 37 into the
• the sensor card 10 fits into the heater block
• microprocessor 40 cycles the temperature of the sensor card 50 and the solution in contact with
• the sensors inside the sensor card 50 through a specific temperature for a specified duration.
• heater block assembly 39 is capable of rapid heating and cooling by, for example, a
• thermoelectric device applying, for example, the Peltier-effect, monitored by a thermistor 41, all
• the sensors connect to the electrode interface 38 which
• analog board 45 where it is converted from analog to digital form, suitable for storage and
• the electrode assembly 10 has a number of edge connectors 36 in a
• formed on the assembly 10 may be connected to microprocessor 40 through the analog board 45.
• the microprocessor 40 is connected to the multiport valve 18 via a valve driver 43 by a line 42
• microprocessor 40 controls the position of the sample arm 5 through arm driver 15, and the
• the electrodes forming part of the assembly make measurements of the parameters of the sample
• microprocessor 40 stores these electrical values. Based upon measurements made during
• the microprocessor 40 effectively creates a calibration curve for each of the measured
• microprocessor 40 is suitably programmed to perform measurement, calculation, storage, and
• tonometered with 27% O 2 , 5% CO 2 , and 68% Helium gas is as follows: pH 7.40 organic buffer;
• membrane for the enzyme sensors contains aqueous solution of Na + , K + , Ca salt; 15 mmol/L of
• compositions of the A and B calibrating solutions are chosen so that for each of the
• the AO calibrating solution is chosen for low level oxygen
• the A and B calibration compositions are prepared by premixing all of the constituents in
• the AO calibration solution is prepared with a slight difference in procedure.
• the salts with the exception of sodium sulfite, /w-phenylenediamine and sodium bicarbonate are
• At least one electropolymerizable monomer is added to at least one of the calibrating
• monomers -phenylenediamine for example, may be included in a calibrating solution at a
• concentration in a range between about 1 to lOOmM, preferably 15mM.
• electropolymerizable monomer may be included in the cartridge 37 in a separate reservoir.
• the calibration solutions must not be packaged at too low a pressure i.e., not below about 625 mm of mercury, because the absorptive capacity of
• the absorptive capacity of the solution may be sufficiently high that it will tend to
• a calibrating solution prepared at a temperature in excess of its
• Calibration Solution A, B and AO are prepared at a temperature above its intended use
• the envelope is first evacuated and then filled with the prepared solution. The bag is then shaken
• the reference solution disposed in prepackaged container 28 is employed in the electrode
• the solution is 1 mol/L potassium nitrate and 1 mmol/L silver nitrate solution.
• solution also contains a surfactant such as Brij 35.
• the solution is packaged in a sealed flexible
• the elecfrode assembly 10 receives
• the electrode assembly 10 in a preferred embodiment consists of a structurally rigid rectangular card 50 of polyvinylchloride having a
• rectangular aluminum (or other suitable material) cover plate 52 adhered to one of its surfaces.
• Cover plate 52 closes off the flow channels 56 fo ⁇ ned in one surface of the card 50 and also acts
• a suitable heating or cooling element e.g., a Peltier-effect device and thermistor 41 to
• a reference solution is introduced to a well 64, formed in the surface
• the reference solution flow line 30 passes through an inclined hole in the well 64.
• the well 64 is connected to the output section 34 of the flow channel 56 through a very thin
• capillary section 66 formed in the surface of the plastic substrate 50 in the same manner as the
• the capillary chamiel 66 is substantially shallower and narrower than the
• main flow channel 56 its cross section is approximately 0.5 sq. mm. Reference fluid pumped
• main flow channel section 56 then flows with it to the waste bag 32.
• the heat plate 52 abuts and forms one wall of the sample
• the heat plate 52 is in contact with the Peltier-effect device of the thermal block
• the thermal block assembly 39 is capable of changing and
• the temperature change and control is monitored by a thermistor 41 and regulated by the microprocessor 40.
• internal digital clock of the microprocessor 40 controls time and can switch on and switch off the
• thermal block assembly 39 according to a preset program.
• microprocessor 40 controls the
• thermal block assembly 39 regulating the temperature setting and the duration of each set
• a pair of gold wires 98 and 100 form electrodes for determining the
• Hct hematocrit
• circuit edge connectors 102 and 104 respectively, also illustrated in FIG. 5.
• the next sensor in the flow channel 56 is the oxygen sensor 70 with a
• Electrode 86 and a potassium sensing elecfrode 90 including an active membrane and a staked
• pH sensing electrode 94 next along the flow channel 56 is a pH sensing electrode 94 also
• FIG. 6 which includes a membrane 148 and a silver wire 87 staked or press-fitted
• the next electrode 93 along the flow channel 56 measures the
• pH electrode 94 pH electrode 94.
• lactate electrode 92 functions by
• lactate oxidase measuring by-products of an enzymatic reaction of lactate oxidase on lactate.
• oxidase present in the enzyme layer oxidizes the lactate producing hydrogen peroxide, which is
• a glucose electrode 91 is the next electrode, which like the lactate
• electrode 92 functions by the detection of hydrogen peroxide produced by an enzymatic reaction
• the enzyme glucose oxidase, specifically oxidizes glucose and produces
• Measurement of creatinine in a blood sample requires two electrodes.
• One electrode One electrode
• concentration of only creatine The concentration of creatinine is determined by subtraction of
• the enzyme layer includes a mixture of three enzymes: creatininase, creatinase and sarcosine oxidase. This enzyme mixture specifically
• the enzyme layer includes a mixture of two enzymes: creatinase
• This enzyme mixture specifically oxidizes only creatine and produces
• the ground 105 illustrated in FIG. 2 is a silver wire inserted through the substrate 50.
• ground serves as a common electric reference point for all electrodes.
• the ground may also serve
• bubbles may form in the reference channel as a result of degassing the reference solution at the
• a printed circuit element 108 also illustrated in FIG.
• Ion-selective membranes of this type which are also known as liquid membranes,
• Polymers for use in the ion-selective membrane of the instant invention include any of the
• hydrophobic natural or synthetic polymers capable of forming thin films of sufficient
• polyurethanes particularly aromatic polyurethanes
• copolymers of polyvinyl chloride particularly aromatic polyurethanes
• the ionophore used in the ion-selective membrane is generally a substance capable of
• the selectivity of the electrode for a particular ion is due to the chemical nature of the
• valinomycin a potassium-selective ionophore
• nonactin dinactin, trinactin
• enniatin group enniatin A, B
• cyclohexadepsipeptides gramicidine, nigericin, dianemycin, nystatin, monensin,
• esters of monensin especially methyl monensin for sodium ion-selective
• concentration of ionophore in the membrane will, of course, vary with the particular
• Ionophore concentrations of between about 0.3 and about 0.5 g/m 2 are prefe ⁇ ed. The ionophore
• the plasticizer provides ion mobility in the membrane and, the presence of a plasticizer is
• the plasticizer must, of course, be compatible with the membrane polymer and be a
• solvent for the ionophore which is also compatible with the polymer may be used. It is also
• the ion plasticizer be substantially non-volatile to provide extended shelf-life for the
• phthalates phthalates, sebacates, aromatic and aliphatic ethers,
• plasticizers include trimellitates, bromophenyl phenyl ether, dimethylphthalate, dibutylphthalate,
• this layer below about 5 mils and preferably about 1 mil.
• the uniformity of thickness of the ion selective membrane plays an important role in the optimum utilization of electrodes of the type described herein.
• the electrodes of the present invention include a support or card 50
• the support may comprise ceramic, wood, glass, metal,
• support carrying the overlying electrode components must be inert; i.e., it does not interfere with
• composition of the support must withstand elevated
• the support According to a highly prefe ⁇ ed embodiment of the present invention, the support
• polymeric support may be of any suitable thickness typically from about 20-200 mils. Similarly thin layers or surfaces of other materials mentioned above could be used. Methods for the formation of such layers are well known in the art.
• An enzyme sensor comprises a three-electrode system including a working, reference and counter electrode.
• the working electrode includes a composite membrane that is deposited on a surface in contact with a conductive wire, a platinum wire for example.
• the composite membrane comprises two or more layers, including a enzyme layer and an inner interference rejection membrane, for example.
• the sensor fabrication may be based on solvent casting techniques well known in the art.
• the thickness of the layers can be controlled by dispensing precise volumes of solutes found in the layers.
• the polymeric membrane that comprises an inner interference rejection membrane, described in detail below, is formed onto the wire electrode by electropolymerization of electropolymerizable monomers, as described below.
• an enzyme electrode 59 such as a glucose electrode, is located in the flow channel 56 of the sensor card 50.
• FIG. 3B is an enlarged section of FIG. 3 A.
• the enzyme electrode 59 includes a three layer composite membrane 60 comprising, from the flow channel 56 to the wire 57, an outer membrane 51 adjacent to the flow channel 56, an enzyme layer 53, located between the outer membrane 51 and an inner interference rejection membrane 55 adjacent a wire 57.
• the enzyme electrode 59 contacts the sample as the sample flows along the flow channel 56 and over the outer membrane 51 of the enzyme electrode 59.
• the electrical signal generated by the enzyme electrode 59 is carried by the wire 57 and transfe ⁇ ed to the conductor 61 which is in electrical communication with the electrode assembly
• the outer membrane 51 is a polymeric membrane comprising
• the hydrophobicity of the membrane is determined
• membrane 51 is the concentration in which an optimal balance of diffusion rates of oxygen
• a highly hydrophobic outer membrane may be prefe ⁇ ed because oxygen will
• the outer membrane 51 may have a preferable thickness of 8 to 15 microns and could
• the outer membrane 51 is composed of a blend of polyurethanes with different water
• a typical composition of the outer membrane 51 is 77% aliphatic, polyether-based
• polyurethane with 20% water uptake, 17% aliphatic, polyether-based polyurethane with 60%
• membrane 51 with this composition can be produced by dispensing a volume from a solution of
• the outer membrane 51 which is layered directly onto and in
• enzyme 49 is embedded, to degradatory proteins or compounds from the sample in channel 56.
• outer membrane 51 prevents diffusion of the enzyme 49 out of the enzyme layer 53.
• the outer membrane 51 also functions to control the rate of diffusion of analyte (e.g. glucose,
• the enzyme layer 53 of the glucose or lactate sensor includes at least at
• the enzyme layer 53 participates that is stabilized in the matrix of the enzyme layer 53.
• the enzyme layer 53 participates that is stabilized in the matrix of the enzyme layer 53.
• enzyme 49 includes at least one protein with enzymatic activity. In other embodiments, enzyme
• 49 includes a mixture of several enzymes, proteins and stabilizers, for example.
• lactate oxidase are embedded in the enzyme layer 53 and create an electrode 91 and 92
• electrode 91 includes glutaraldehyde and glucose oxidase in the enzyme layer 53.
• the glucose electrode 91 may include 0.10 g of glutaraldehyde per gram of glucose
• the lactate electrode 92 includes at least glutaraldehyde
• bovine serum albumin a enzyme stablizer such as, for example, polyethyleneimine and lactate
• the lactate electrode 92 includes 45% lactate oxidase by weight, 45% bovine serum albumin by weight, 5% polyethylenimine (an enzyme
• lactate oxidase and bovine serum albumin can vary.
• the enzyme layer can vary from 1 to 20, and the weight percent of glutaraldehyde can vary from 1
• enzymes stabilizers include but are not limited to polyionic compounds such as
• polypropyleneimine poly(N-vinylimidazole), polyallylamine, polyvinylpiridme, polyvinylpyroUidone, polylysine, protamine and their derivatives.
• enzyme layer 53 includes a mixture of
• creatinine electrode 116 includes a mixture of 5% creatininase
• vinylimidazole (an enzyme stabilizer) by weight and 5% glutaraldehyde by weight, for example.
• the weight fractions of creatinase and sarcosine oxidase in the creatine electrode can vary.
• the weight percent of poly(N-vinylimidazole) in creatinine and creatine electrodes can vary, for
• creatine electrodes can also vary, for example, from 1% to 10%.
• poly(N-vinylimidazole) can also be used for stabilizing the enzyme mixture.
• polyionic compounds include but are not limited to polyethylemmine, polypropyleneimine,
• polyallylamine polyvinylpiridme, polyvinylpyroUidone, polylysine, protamine, and their
• layer 53 consists of a cross-linked matrix of enzymes, stabilizers such as polyethylenimine or
• enzymes, stabilizers, and other protein molecules is accomplished with, for example,
• diisocyanato, 1,2,7,8-diepoxyoctane and 1,2,9,10-diepoxydecane, both diepoxides, can also be
• proteins in the enzyme matrix can significantly extend the shelf-life and the use-life of the enzyme matrix
• enzyme layer 53 includes a mixture of several enzymes, proteins, but lacks an enzyme
• the creatinine electrode 116 includes a mixture of 30%
• the creatine electrode 118 includes a mixture of 45% creatinase
• the enzyme layer 53 45% sarcosine oxidase and 10% glutaraldehyde (percentages by weight).
• the thickness in the range of 1 to 10 microns, preferably 2-5 microns measured from the
• the enzyme electrode 59 also includes an inner
• interference rejection membrane 55 which is a restorable polymeric membrane in close contact to
• the inner interference rejection membrane 55 may be formed by the polymerization
• Suitable electropolymerizable monomers include
• membrane 55 which is typically less than a micron thick, insulates or protects the wire 57 from compounds in the sample, specifically oxidizable compounds, that interfere with the proper
• the polymeric membrane comprising the
• inner interference rejection membrane 55 is formed by the application of an electrical potential to
• Hydrogen peroxide which is generated from activity of the enzyme of the enzyme electrode on a
• sample larger than hydrogen peroxide, such as acetaminophen, ascorbic acid, uric acid, cysteine
• 55 may be regenerated on a repeated basis to restore its function. Following repeated exposure to
• the inner interference rejection membrane 55 is degraded or fouled by compounds
• the wire 57 from interfering compounds present in the analytical sample e.g., ascorbic acid
• an electropolymerizable monomer can be combined with a calibration solution
• system 8 illustrated in FIG. 1 for example, for use in the repolymerization and restoration of the
• monomer-containing AO solution is pumped from a prepackaged container and passed through
• the electrical potential polymerizes the monomer
• interference rejection membrane 55 can be employed to account for different situations.
• sensor system 8 illustrated in FIG. 1 is applied to the wire 57 in the range of 0.1 to 0.8 V versus
• An oxygen sensor comprises a three electrode system including a working electrode, a
• working electrode 70 comprises a platinum wire 74 that is fixed in the center of an insulative
• the board 50 preferably has a thickness of approximately 40 mils while the board 50 may have a thickness of
• the diameter of the glass disk is preferably about 100 mils.
• a number of the glass disks with the embedded platinum wires are prepared by inserting
• given axial thickness are sliced off, by a power saw, for example.
• the glass disk is practically impervious to oxygen whereas the polyvinylchloride of the
• the glass disk thus protects the platinum electrode 74 from the
• the two membranes 120 and 122 on the glass disk protect the platinum wire 74 from
• membrane 120 is a hydrogel based on methacrylic esters that is covalently bonded to the glass
• the membrane 122 underneath the 120 covers only the area around the platinum wire and
• the composite membrane 60 including 120 and 122 provides a
• hydrogel that is employed is based on methacrylic esters, although hydrogels not based on esters
• the monomer such as hydroxyethyl
• methacrylate or hydroxypropyl mefhacrylate for example, is copolymerized with a cross-linker
• the cross-linking reaction can be initiated by a photoinitiator such as
• a solvent such as ethylene glycol or water can be used to dilute
• the surface of the glass disk is silinized with hexamethyldisilazane and functionalized
• methacrylic groups by reacting with trimethoxysilyl propyl methacrylate.
• the disk is exposed to a broad band UV light for 5 min to photopolymerized
• the glass disk with the composite membrane on one side of it is embedded in a recessed
• electrodes form the working electrode allows for smaller size of the working electrode and
• processor 40 which lessened potential serves to reduce any oxygen reaching its end and thereby
• Electrodes H. Potassium, Sodium and Calcium Sensing Electrodes
• the electrodes best illustrated generally in FIG. 2, connecting the silver wires 78, 86, 90,
• FIG. 93, and 94 which sense Na, Ca, potassium, pCU 2 and pH activities, respectively, are similar in construction. The difference is in the composition of the membrane layers.
• a typical ion- selective electrode is illustrated in FIG 6. Each has a bead or an inner salt layer 152, which upon hydration forms the inner solution layer. This layer is in contact with the thin film of silver/silver chloride layer 154 obtained by anodization of the top of the silver wires.
• the outer layer 148 is essentially the polymeric ion-selective membrane layer.
• This layer is formed over the dried salt residue of the inner layer in a shallow well 150 as a dry residue remaining after the solvent removal from a matrix of a permeable hydrophobic membrane forming solution such as a solution containing polyvinylchloride, a plasticizer, an appropriate ion-sensing active ingredient and a borate salt.
• a permeable hydrophobic membrane forming solution such as a solution containing polyvinylchloride, a plasticizer, an appropriate ion-sensing active ingredient and a borate salt.
• the outer membrane is applied as a solution, typically in Tetrahydrofuran (THF) in a small droplet. Once the solvent evaporates, the membrane is formed and is bonded to the plastic card.
• THF Tetrahydrofuran
• the ion-selective active ingredient may be tridodecylamine (TDD A) or a suitable pH sensing component.
• a monocyclic antibiotic such as valinomycin may be used as the active ingredient.
• the calcium electrode employs a calcium ion-selective sensing component as its active ingredient such as (-)- (R,R)-N,N'-(Bis(l l-ethoxycarbonyl)undecyl)-N,N'-4,5-tetramethyl-3,6-dioxaoctanediamide; Diethyl N,N'-[(4R,5R)-4,5-dimethyl-l,8-dioxo-3,6-dioxaoctamethylene]-bis(12-methylamino- dodecanoate) or other suitable calcium sensitive selective substance.
• the sodium electrode employs methyl monensin ester or any other suitable sodium sensitive active ingredient.
• the sodium, potassium and calcium electrodes use a buffer salt like MES (2-[N- morpholinojethanesulphonic acid) along with the respective chloride salts for their inner solution. pH and pCO 2 electrodes share the same outer layers, while their inner layers differ
• the internal layer for pH uses a strong buffer, for example, MES buffer, while that
• the CO 2 sensor is a combination of CO 2 and pH electrodes working together. In function
• aqueous salt solution usually a
• Hydration from a dry state can be accelerated by soaking the sensors in an electrolyte solution, such as the calibrating solutions described above,
• the sensors are soaked in calibrating solution B at a temperature between 55°C to
• the sensors are soaked in a calibrating
• the hematocrit (Hct) measurement is made through a measurement of resistivity between
• the sensor operates by measuring the resistivity of the solution or blood
• Hematocrit is calculated as a function of resistivity using
• composite membrane 60 to retain residual concentrations of substrate from the sample
• a polarization pulse may be applied by an electrical source to the wire 57 after each
• hydrogen peroxide a product of the reaction of the enzyme and the analyte from the operation of the electrode 59, is an example of an interfering agent.
• the analytes such as glucose and lactate
• step includes restoration of the integrity and proper functioning of the inner interference rejection
• the electropolymerizable monomers are in solution in the AO
• the electropolymerizable monomers contact the enzyme electrode 59 at the outer polymeric
• reference electrode for is applied to the wire 57 for 3 minutes, for example, causing the
• the reference solution fills the well 64 where it
• the reference solution is essentially a hypertonic solution of potassium nitrate
• electrode 106 constitutes a stable potential liquid junction formed between the reference
• the reference solution is of high density and under pumping force must flow upward against gravity to the outlet.
• the reference solution remains stationary in the reference well 64
• the capillary tube 66 due to the density gradient, acts as a one way
• valve allowing pumped reference solution to pass upwardly through the capillary but preventing
• the heater block assembly 39 includes a thermoelectric device
• thermistor 41 an aluminum block featuring two aluminum shells 220a, 220b, electrode
• the aluminum block houses a sensor card 10 when the cartridge with the sensor card is inserted
• the aluminum heater block assembly 39 includes two aluminum
• shells 220a, 220b which together form a socket 222 into which a sensor card 10 (not shown) can
• a cable 226 connects the electrical connectors from the sensor
• a printed circuit board to a microprocessor 40 through an analog board 45 (See FIG. 1).
• a printed circuit board to a microprocessor 40 through an analog board 45 (See FIG. 1).
• Printed circuit boards within this heater block assembly contain post amplifiers that amplify
• the output of the sensors are analog signals.
• analog signals are converted to digital signals via an analog to digital converter, and the digital
• a thermistor 41 is located as illustrated in FIG. 7C. Extending from
• thermistor 41 are electrical connections 229, 229' that connect the thermistor 41 to a
• microprocessor 40 The microprocessor 40.
• thermoelectric device 230 illustrated in FIG. 7D is positioned. Thermoelectric devices in the
• heater block assembly may use, for example, the Peltier-effect, to heat and cool the aluminum
• Electrical leads 231, 231' supply programmed electrical cu ⁇ ent controlled by a
• thermoelectric device 230 The direction and duration of cu ⁇ ent is
• thermoelectric device 230 controlled by the microprocessor 40 and determines whether the thermoelectric device 230
• overlying the aluminum shell 220b is in a warming or cooling mode.
• aluminum shell 220b is measured by thermistor 41 which transmits signals to microprocessor 40.
• Microprocessor 40 is programmed to transmit electrical signals to the thermoelectric device,
• the metal plate 220b is cooled and the cooling effect is transmitted to the
• thermoelectric device 230 Refe ⁇ ing to FIGS. 7D and 7E, the external surface 233 of the thermoelectric device 230
• metal plate 234 is in contact with a metal plate 234.
• the external surface 235 of metal plate 234 is in contact
• thermoelectric thermoelectric
• thermoelectric device 230 thermoelectric device 230
• electrical leads 231, 231' from the thermoelectric device 230 to the microprocessor 40 is
• the temperature for a sensor In a prefe ⁇ ed embodiment of the heater block assembly 39, the temperature for a sensor
• cartridge can be increased from about 37°C to about 60°C to 65°C in one minute, maintained at
• valve 18 is controlled to direct one of the calibration solutions
• calibration solution B for example, calibration solution B, into the sensor assembly so it entirely fills the flow channel.
• the pump is then stopped for a period of 10-30 minutes, preferably 12-15 minutes during which
• the dry chemical sensor electrodes are hydrated by thermal cycling, for example, from 37°C to
• the dry chemical electrode sensor assembly 10 is
• thermoelectric microprocessor 40 reverses cu ⁇ ent flow through the thermoelectric
• thermal plate 52 are cooled to 37°C.
• the temperature, controlled by the microprocessor 40, is maintained at 37°C for the life of the cartridge 37. After hydration of the sensors, the
• conditioning cycle of the enzyme electrodes 59 starts by pumping the AO solution 23 to the
• the polarization potential of the enzyme electrodes 59 is elevated from 0.25 to 0.5 N versus the
• the low oxygen level is also calibrated.
• the rinse cycle the low oxygen level is also calibrated.

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